Monoclonal antibodies have been used within the medical field, at least, for many years, particularly as a means to permit binding to specific antigens within an organism. In such a manner, such antibodies can be created to target certain types of cells to promote any type of response, primarily deactivation, detection, and/or purification of substances.
Generally speaking, monoclonal antibodies are structures that are substantially the same in composition and form that are made by identical immune cells, being, in essence, and as their name implies, clones of a unique parent cell. Thus, these antibodies exhibit monovalent affinity for the same target epitope, permitting a rather large-scale (at a microscopic level) potential platform to effectively treat certain infectious (or other like) substances within an organism. As such, it has been realized that it is possible to produce monoclonal antibodies that can be engineered to actually bind to any given substance. Thus, monoclonal antibodies have been long utilized as important tools to provide effective targeted treatments, medically and otherwise, for undesirable in vivo substances.
The general method of monoclonal antibody production is well known. However, in order to properly provide a suitable medicament, etc., including such biochemical structures, one must first ensure that the antibodies themselves do not include extraneous substances themselves that could prove deleterious to the target organism (or even possibly counteract the effectiveness of the antibodies during application). For instance, the potential for harmful mammalian virus transfer through the utilization of animal cells (such as CHO, NSO, Per-C6, as examples) for such a purpose, as well as the relatively large cost of such testing protocols certainly give pause as to the continued implementation of such mammalian-based monoclonal antibody harvesting. Such residual virus particles, herein defined as materials that are capable of initiating an infection within a mammalian cell, require a number of actions to ensure utility of such antibodies subsequent to actual production. For instance, inactivation of the virus strains themselves are required to provide reliable results (since total removal from such produced materials may not be achieved or, alternatively, active viruses may deleterious effect the production method itself). Such necessary inactivation and removal steps are to the overall complexity and costs of mammalian-based antibody production schemes. Avoidance of these added steps and potential problems are of great importance to allow for more streamlined antibody production capabilities, certainly.
Otherwise, there still exists the necessity to purify such resultant structures, leaving as close to just the desired specific monoclonal antibody constructs alone. There are a wide variety of purification techniques that have been developed for this purpose, with each procedure seemingly directed to highly specific antibody structures, as opposed to the potential for purification processes for a wide variety of antibodies. Although the specific methods that have been employed may be effective for such individualized antibody structures, unfortunately these unique procedures are not cost-effective as there are very few locations and companies that center on a single monoclonal antibody production scheme. To the contrary, typically multiple antibody formulations are produced at the same place, thus requiring not only divergent starting materials and controls in that respect, but also the required undertaking of shifting purifying methods at great expense in order to target such specific end results. There thus exists a significant need to provide not only an effective all-encompassing purification method for monoclonal antibody production purposes, but also a need to provide such a purification platform that accords a reliable method in that respect that guarantees, to a certain degree, at least, that the resultant antibodies will exhibit optimal activity levels for their intended use. To date, such a one-size-fits-all approach for antibody production and purification has been natively absent within the biochemical and medical fields.
Certainly, as alluded to above, there have been a wide variety of general purification methods for antibodies. However, again, such techniques are not directed to the potential to provide an overarching purification platform for different types and potentially different classes of monoclonal antibodies. Additionally, many such targeted purification schemes are developed to achieve such ends with minimal processing steps, rather than from a view that standardized procedures may accord greater efficiencies in the long run. As well, these specific purification protocols require the utilization of different reagents, buffers, etc., compounds and formulations that further necessitate additional testing and regulatory compliance if involving antibodies for human ingestion/introduction at some point. Thus, although such “quick” methods may provide effective purification results for specific monoclonal antibody products, in actuality, the overall costs to achieve not just development success, but regulatory compliance, ultimately, militates against such actions.
Additionally, traditional antibody production methods have typically involved a specific fingerprint process for each specific type of compound involved. In essence, a targeted antibody has required a specific production and purification protocol that concerns optimum levels for that specific antibody alone. Thus, if a new antibody were produced, or at least developmental activities were devoted to a specific type or types, in the past the overall provision for such an antibody required a narrow and specific method to generate a threshold amount for testing and ultimate implementation purposes. Such unique fingerprint steps undertaken for antibody generation thus limited efforts in terms of scalability; the requirement that specific methodologies be followed with little to no deviation led to higher potentials for compromised production batches (slight modifications from set protocols could lead to failure in terms of effective results). Thus, there exists a definite need to provide a more uniform, if not a one-size-fits-all approach, to antibody production methods. To date, such has simply not been made available, whether in terms of mammalian-based processes or otherwise.
Of particular interest and appeal in terms of the utilization of monoclonal antibodies (mAbs) for numerous therapeutic and prophylactic treatments (for mammalian subjects, at least) are the potency, specificity, and safety profile of these materials. However, as alluded to above, there are serious challenges involved in the production and development of new mAb products, particularly as it concerns reliable and consistent manufacturing procedures and results. For example, the potential growth and spread of diseases, whether through natural or human-inflicted (e.g., WMD) consequences requires scalability for quick and effective supply of medical treatments utilizing such platforms. In essence, with the number of diseases growing, rather than shrinking, worldwide, larger quantities of mAbs are necessary to meet expected and unexpected disease activities in order to provide for disease protection and treatment. Even moderate production levels may be sufficient for individuals that are subjected to locations prone to exposure to viruses and other weapon-based (for instance) situations, particularly in at-risk areas or for clinical trial applications. Reliability and efficiency, though, will still be paramount in order to properly accord the needed responses and treatments in these instances. It does still remain, however, that extremely high levels of reliable and effective production of proper medical treatments based on mAbs platforms will be necessary to ensure product to address broad disease application or broad civilian exposure in the case of WMD release or even during unexpected virus spread.
Again, as alluded to above, typical mAbs production methods involve mammalian cell reactors. Certainly, such an approach has been successfully employed for predicted supply requirements involving expected disease outbreaks, thus allowing for long timelines for scale-up to meet the overall supply needed for such large-scale treatments. Unfortunately, these types of mammalian cultures, whether small- or large-scale in effect, are not well suited for rapid response and varying scale production. Capital requirements associated with cell growth, space requirements, and even use amortization are rather expensive and the costs for such expenses are rather difficult to achieve. Upstream facilities and slow product turnaround cycles for mammalian based production processes are overly expensive and the lack of definitive understanding (even with expected outbreaks) as to the actual need for such resultant treatment products (vaccines, etc.) has hampered attempts to receive suitable funding (in excess of $500M, typically) for such facilities. Additionally, the long-term development process for new mAb products, particularly in terms of response to outbreaks of broader strains and/or new virus species, and the uncertainty surrounding such possible situations, has not led to any further incentives for investment in updated facilities and/or production processes. Cell line optimization, process adaptation, and requisite scale up requirements, at least, can lead to long-duration monoclonal antibody-based construct development (18-24 hours or longer, for instance), thus not only increasing the time and resources needed for such activities, but the uncertainty in terms of actual achievement of suitable treatments after such a time has passed. Furthermore, the prior mammalian-based mAbs production methods (e.g., within CHO or NSO cells) exhibit suspect and/or insufficient antibody dependent cellular cytotoxicity (ADCC) activity to potently counter rapidly replicating and pervasive pathogens (such as Ebola, for instance). Such ADCC is primarily due core fucose residues present on N-glycans thereby reducing the affinity of such compounds to the FcγRIII receptor responsible for ADCC signaling. Thus, there exists a need to undertake either expensive and uncertain glycan engineering for such mammalian-based products, or the payment of potentially high royalty rates to utilize other processes merely to attempt to modify these base structures sufficiently to ensure overall effectiveness and safety for mammalian treatment subjects. These “extra” significant production costs thus contribute to already high levels that, so far, have led to the majority of research investment to avoid certain niche clinical applications and suspect WMD challenges. These limitations thus demand new, more scalable, responsive and efficacious production strategy.
As it is, the current applications followed within the monoclonal antibody industry have, again, been primarily mammalian-based and, as alluded to above, also concern individualized purification procedures for specific antibody structures. Indeed, typically it requires up to 9 months to optimize a mammalian cell line for monoclonal antibody production. The steps may differ for different approaches, but they require the introduction of at least two different gene constructs for expression into a cell, including genes encoding the two antibody chains plus non-antibiotic selectable markers. Such genes are transfected into cells separately or jointly, and then selected for cell lines that contain both gene constructs and further express a fully assembled, two-chain, monoclonal antibody product. Following selection of expressing lines, the resultant cell lines must be compared for production of monoclonal antibody productivity and often undergo further selection for increased antibody production through gene duplication strategies using methotrexate selection that identifies cells with the highest number of dihydrofolate reductase or other selectable marker genes. These methods require analysis of many separate production cell lines and detailed screening for production capability as measured by amount of antibody per cell, typically, in a culture, for example. Following selection of optimally expressing cell lines, the lines must be optimized for culture conditions, growth characteristics, buffers, nutrients and other variables as well. The overall development process required to produce an antibody in mammalian cells is thus, as alluded to above, rather complicated to manage due to many cell lines to compare, optimize and stabilize, at least. Management of these variables of cell and production conditions requires time, scale, and expense to achieve a final, optimized cell line, too. Further, mammalian cell production requires sequential scaling of cultures from a single Master or Working Cell Bank of 1 mL or similar volume, to 10 mL, 100 mL, 1,000 mL all to subsequently seed the next sequential volume production container. Each seeding process puts cultures at risk for contamination with advantageous agents due to length of culture and the seeding process. The seeding and scale up process for full volume production takes time and a high level of expertise before production occurs. The requirement of quick-time production, in reaction to, for instance, WMD threats, unforeseen disease pandemics, and thus the need for multiple antibodies in a single product, and, furthermore the parallel comparisons of antibodies in such situations, are made very difficult due to the time, repeatable processing, and overall complexity needed to derive an optimal expression clone for each antibody, as well as the scalable requirements of sequential seeding for full volume production. Although many varied purification systems have been developed for mammalian cells (Kelley, MAbs. 2009 September-October; 1(5): 443-452; Shukla, et al., Journal of Chromatography B, 848 (2007) 28-39; Shukla, et al., 2010. Trends in Biotechnology Vol. 28 No. 5. pp. 253-261; Liu et al., 2010. MAbs. 2010 September-October; 2(5): 480-499.), each requires virus inactivation and virus filtration or removal steps that are not required for plant-based systems since they lack viruses that infected mammalian cells.
Thus, to avoid the limitations and potential pitfalls of mammalian-based mAbs production methods and products, there exists a significant need to provide an effective standardized plant-based antibody production and purification method that avoids the complexity of the time consuming process to derive optimal cell lines, optimize culture conditions, manage complex processes for scale up production just to achieve a single antibody and virus inactivation and removal steps, let alone a number of candidates required to optimize a product that may require multi-product content or different product comparison for protection or therapy against a WMD threat or another type of disease (Whaley et al., 2011. Human Vaccines 7:3, 349-356.). Standardized plant-antibody production methodologies are typically dependent of generation of transgenic plant lines that require 6-9 months to derive, and up to three years to generate sufficient seed for full-scale production. The utilization of virus vectors shortens such production time lines (Whaley et al., 2011), but each production process followed in such instances is typically conducted much like mammalian cell production activities (basically undertaking the utilization of highly tailored production and purification processes for each antibody (Ko and Koprowski, Virus Research 111 (2005) 93-100; Jain et al., 2011; Asian journal of Pharmacy and Life Science, Vol. 1(1), January-March and references therein), which thus requires high specificity and, as noted above, increased chances of off-quality batches). The trial and error process to optimize expression processes, production conditions, and purification procedures in such traditional method are time-consuming and expensive. Furthermore, although these highly optimized and tailored processes may yield optimal production levels, the time and resources required, as well as the overall complexity of such processes to produce therapies involving more than a single antibody or rapid product production required for WMD and other like threats, are far too high for economical and efficient operations. In such production methods, there exists a significant need, therefore, to permit repetitive utilization of regulatory compliant formulations, buffers, etc., while still effectuating an acceptable purification result, and all through the reliance upon a plant-based resource. To date, however, there is lacking any such method within the monoclonal antibody production/purification industry.